Increased fucosylation and reduced branching of serum glycoprotein N-glycans in all known subtypes of congenital disorder of glycosylation I

The N-glycans present on the total mixture of serum glycoproteins (serum N-glycome) were analyzed in 24 subjects with congenital disorder of glycosylation type I (CDG-I) and 7 healthy, age-matched individuals. No new N-glycan structures were observed in the sera of CDG-I patients as compared with normal sera. However, we observed in all subtypes a significantly increased degree of core alpha-1,6-fucosylation of the biantennary glycans as compared to normal, as well as a significant decrease in the amount of triantennary glycans. These serum N-glycome changes appear to be a milder manifestation of some of the changes observed in adult liver cirrhosis patients, which is compatible with the reported steatosis and fibrosis in CDG-I patients. In the CDG-Ia subgroup, the extent of the serum N-glycome changes correlates with the aberration of the serum transferrin isoelectric focusing pattern, which measures the severity of the lack of entire N-glycan chains (primary consequence of CDG-I) in the liver and is the standard diagnostic test for this category of inherited diseases.     

Analysis of the site-specific N-glycosylation of beta1,6 N-acetylglucosaminyltransferase V

N-acetylglucosaminyltransferase V (GnT-V) catalyzes the addition of a beta1,6-linked GlcNAc to the alpha1,6 mannose of the trimannosyl core to form tri- and tetraantennary N-glycans and contains six putative N-linked sites. We used mass spectrometry techniques combined with exoglycosidase digestions of recombinant human GnT-V expressed in CHO cells, to identify its N-glycan structures and their sites of expression. Release of N-glycans by PNGase F treatment, followed by analysis of the permethylated glycans using MALDI-TOF MS, indicated a range of complex glycans from bi- to tetraantennary species. Mapping of the glycosylation sites was performed by enriching for trypsin-digested glycopeptides, followed by analysis of each fraction with Q-TOF MS. Predicted tryptic glycopeptides were identified by comparisons of theoretical masses of peptides with various glycan masses to the masses of the glycopeptides determined experimentally. Of the three putative glycosylation sites in the catalytic region, peptides containing sites Asn 334, 433, and 447 were identified as being N-glycosylated. Asn 334 is glycosylated with only a biantennary structure with one or two terminating sialic acids. Sites Asn 433 and 447 both contain structures that range from biantennary with two sialic acids to tetraantennary terminating with four sialic acids. The predominant glycan species found on both of these sites is a triantennary with three sialic acids. The appearance of only biantennary glycans at site Asn 433, coupled with the appearance of more highly branched structures at Asn 334 and 447, demonstrates that biantennary acceptors present at different sites on the same protein during biosynthesis can differ in their accessibility for branching by GnT-V.     

Expression of functional human transferrin in stably transfected Drosophila S2 cells

Human transferrin (hTf) is a serum glycoprotein involved in Fe3+ transport. Here, a plasmid encoding the hTf gene fused with a hexahistidine (His6) epitope tag under Drosophila metallothionein promoter (pMT) was stably transfected into Drosophila melanogaster S2 cells as a nonlytic plasmid-based system. Following 3 days of copper sulfate induction, transfected S2 cells were found to secrete hTf into serum-free culture medium at a competitively high expression level of 40.8 microg/mL, producing 6.8 microg/mL/day in a 150-mL spinner flask culture. Purification of secreted recombinant hTf using immobilized metal affinity chromatography (IMAC) yielded 95.5% pure recombinant hTf with a recovery of 32%. According to MALDI-TOF mass spectrometry analysis, purified S2 cell-derived His6-tagged recombinant hTf had a molecular weight (76.4 kDa) smaller than that of native apo-hTf (78.0 kDa). 2-Dimensional gel electrophoresis patterns showed recombinant hTf had a simpler and less acidic profile compared to that of native hTf. These data suggest recombinant hTf was incompletely (noncomplex) glycosylated and lacked sialic acids on N-glycans. However, this difference in N-glycan structure compared to native hTf had no effect on the iron-binding activity of recombinant hTf. The present data show that a plasmid-based stable transfection S2 cell system can be successfully employed as an alternative for producing secreted functional recombinant hTf.     

Rat aglycotransferrin and human monoglycotransferrin: production and metabolic properties

Rat transferrin (rTf), containing one complex glycan, and human transferrin (hTf), containing two complex glycans, were treated with peptide:N-glycosidase F (PNGase) under nondenaturing conditions. Apo rTf with a nonfucosylated standard biantennary glycan, but not its diferric counterpart, yielded satisfactory amounts (approximately 55% in 7 h) of aglyco Tf (AgTf). The presence of a chitobiose core fucose reduced yields to approximately 30%, whereas an additional NeuAc on the GlcNAc in the Man(alpha 1-3) branch had no adverse effect. Triton X-100 impaired deglycosylation. The main product (approximately 65%) obtained from apo hTf was monoglyco Tf (MgTf). Analysis of the cyanogen bromide fragments of MgTf revealed that PNGase did not discriminate between the two glycosylation sites of hTf. A negligible portion (2-4%) of AgTf, that was also obtained during the reaction, probably resulted from PNGase action on denatured hTf molecules. Modified Tfs were separated by affinity chromatography, radiolabeled, mixed with another preparation of interest, and injected in rats. Total-body radiation measurements showed that the half-life of rat AgTf was 19-20% shorter than that of rTf but 9% longer than that of asialo Tf. This suggests that close to 76% of the change in the degradative rate observed after desialylating rTf is referable to charge loss rather than the exposure of Gal residues. Human MgTf was catabolized by rats 8-9% faster than the parent protein, while human AgTf behaved in vivo like a denatured protein. It is concluded that sialyl carboxyls are a codeterminant of the normal lifetime of transferrins.     

Structural study of the N-glycans of intercellular adhesion molecule-5 (telencephalin)

Intercellular adhesion molecule-5 (ICAM-5, telencephalin) is a dendritically polarized membrane glycoprotein expressed in tissues distinct from those expressing other ICAMs. Here, we determined the N-glycan structure of ICAM-5 purified from adult rat brain and compared it with that of other ICAMs. N-glycans were released by N-glycosidase F digestion and labeled with p-amino benzoic octylester (ABOE). ABOE-labeled glycans were analyzed by high performance liquid chromatography (HPLC) and mass spectrometry. The N-glycans obtained from rat brain ICAM-5 consisted of approximately 85% neutral, 10.2% sialylated-only, 2.8% sulfated-only, and 1.2% sialylated and sulfated glycans. Compared with the N-glycan structures of human ICAM-1 expressed in CHO cells, HEK cells, or mouse myeloma cells and ICAM-3 isolated from human T-cells, rat brain ICAM-5 had less highly branched glycans, sialylated glycans, and N-acetyllactosamine structures. In contrast, high-mannose-type N-glycans and Lewis X were more commonly found in rat brain ICAM-5 than in human ICAM-1 expressed in CHO cells, HEK cells, or mouse myeloma cells and ICAM-3 isolated from human T-cells. In addition, sulfated glycans contained GlcNAc 6-O-sulfate on the non-reducing terminal side. Our data will be important for the elucidation of the roles of the N-glycans expressed in neural cells, including those present on ICAM-5.     

Man5GlcNAc2哺乳动物甘露糖型糖蛋白的毕赤酵母表达系统构建

蛋白的糖基化对蛋白的活性、高级结构及功能都有重要的影响。酵母表达的糖蛋白不同于哺乳动物表达的杂合型或复杂型糖蛋白,而是高甘露糖型或过度甘露糖化糖蛋白。在前期成功敲除毕赤酵母α-1,6-甘露糖转移酶(Och1p)基因、阻断毕赤酵母过度糖基化,获得毕赤酵母过度糖基化缺陷菌株GJK01 (ura3、och1) 的基础上,通过表达不同物种来源的α-1,2-甘露糖苷酶I (MDSI) 的活性区与酵母自身定位信号的融合蛋白,并通过DSA-FACE (基于DNA测序仪的荧光辅助糖电泳) 分析筛选报告蛋白HSA/GM-CSF (人血清白蛋白与粒细胞-巨噬细胞集落刺激因子融合蛋白) 的糖基结构,发现当编码酿酒酵母α-1,2-甘露糖苷酶 (MnsI) 基因的内质网定位信号与带有完整C-端催化区的拟南芥MDSI基因融合表达时,毕赤酵母工程菌株能够合成Man5GlcNAc2哺乳动物甘露糖型糖蛋白。这为在酵母体内合成类似于哺乳动物杂合型或复杂型糖基化修饰的糖蛋白奠定了基础。     

Site-specific N-glycosylation identification of recombinant human lectin-like oxidized low density lipoprotein receptor-1 (LOX-1)

Human LOX-1/OLR 1 plays a key role in atherogenesis and endothelial dysfunction. The N-glycosylation of LOX-1 has been shown to affect its biological functions in vivo and modulate the pathogenesis of atherosclerosis. However, the N-glycosylation pattern of LOX-1 has not been described yet. The present study was aimed at elucidating the N-glycosylation of recombinant human LOX-1 with regard to N-glycan profile and N-glycosylation sites. Here, an approach using nonspecific protease (Pronase E) digestion followed by MALDI-QIT-TOF MS and multistage MS (MS(3)) analysis is explored to obtain site-specific N-glycosylation information of recombinant human LOX-1, in combination with glycan structure confirmation through characterizing released glycans using tandem MS. The results reveal that N-glycans structures as well as their corresponding attached site of LOX-1 can be identified simultaneously by direct MS analysis of glycopeptides from non-specific protease digestion. With this approach, one potential glycosylation site of recombinant human LOX-1 on Asn(139) is readily identified and found to carry heterogeneous complex type N-glycans. In addition, manual annotation of multistage MS data utilizing diagnostic ions, which were found to be particularly useful in defining the structure of glycopeptides and glycans was addressed for proper spectra interpretation. The findings described herein will shed new light on further research of the structure-function relationships of LOX-1?N-glycan.     

Characterization of the N-linked glycans of Giardia intestinalis

This article reports the first rigorous evidence for the existence of N-glycans in Giardia intestinalis, a parasite that is a widespread human pathogen, being a major cause of enteric disease in the world. Excreted/secreted molecules of G. intestinalis are known to stimulate the immune system. Structural strategies based on MALDI and electrospray mass spectrometry were employed to examine the excreted/secreted molecules for their N-glycan content. These revealed that the major oligosaccharides released by peptide N-glycosidase F are complex-type structures and correspond to bi-, and triantennary structures without core (alpha1,6) fucosylation. The major nonreducing epitopes in these complex-type glycans are: Galbeta1-4GlcNAc (LacNAc) and NeuAc alpha2-6Galbeta1-4GlcNAc (sialylated LacNAc).     

Structural analysis of free N-glycans occurring in soybean seedlings and dry seeds

The structures of unconjugated or free N-glycans in stems of soybean seedlings and dry seeds have been identified. The free N-glycans were extracted from the stems of seedlings or defatted dry seeds. After desalting by two kinds of ion-exchange chromatography and a gel filtration, the free N-glycans were coupled with 2-aminopyridine. The resulting fluorescence-labeled (PA-) N-glycans were purified by gel filtration, Con A affinity chromatography, reverse-phase HPLC, and size-fractionation HPLC. The structures of the PA-sugar chains purified were analyzed by the combination of two-dimensional sugar chain mapping, jack bean alpha-mannosidase digestion, alpha-1,2-mannosidase digestions, partial acetolysis, and ESI-MS/MS. The free N-glycan structures found showed that two categories of free N-glycans occur in the stems of soybean seedlings. One is a high-mannose type structure having one GlcNAc residue at the reducing end (Man 9 approximately 5 GlcNAc1, 93%), that would be derived by endo-GM (Kimura, Y. et al., Biochim. Biophys. Acta, 1381, 27-36 (1998)). The other small component is a xylose-containing type one having two GlcNAc residues at the reducing end (Man3Xyl1GlcNAc2, 7%), which would be derived by PNGase-GM (Kimura, Y. and Ohno, A., Biosci. Biotechnol. Biochem., 62, 412-418 (1998)). The detailed structural analysis of free glycans showed that high-mannose type free N-glycans (Man 9 approximately 5 GlcNAc1) in the soybean seedlings have a common core structural unit; Manalpha1-6(Man1-3)Manalpha1-6(Manalpha1-3)Ma nbeta1-4GlcNAc. Comparing the amount of free N-glycans in the seedling stems and dry seeds, the amount in the stems of seedlings was much higher than that in the dry seeds; approximately 700 pmol per one stem, 8 pmol in one dry seed. This fact suggested that free N-glycans in soybean seedlings could be produced by two kinds of N-glycan releasing enzymes during germination or seedling-development.     

Glycosylation of site-specific glycans of alpha1-acid glycoprotein and alterations in acute and chronic inflammation

BACKGROUND: alpha(1)-Acid glycoprotein (AGP), an acute phase reactant, is extensively glycosylated at five Asn-linked glycosylation sites. In a number of pathophysiological states, including inflammation, rheumatoid arthritis, and cancer, alterations of Asn-linked glycans (N-glycans) have been reported. We investigated alteration of N-glycans at each of glycosylation sites of AGP in the sera of patients with acute and chronic inflammation. METHODS: AGP purified from sera was digested with Glu-C and the liberated glycopeptides were isolated by reverse phase HPLC. N-glycans released with peptide N-glycosidase F and followed by neuraminidase treatment were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. RESULTS: Site-specific differences in branching structures were observed among N-glycosylation sites 1, 3, 4 and 5. Within the sera of patients with acute inflammation, increases in bi-antennary and decreases in tri- and tetra-antennary structures were observed, as well as increases in alpha1,3-fucosylation, at most glycosylation sites. In the sera of patients with chronic inflammation, increased rates of tri-antennary alpha1,3-fucosylation at sites 3 and 4 and tetra-antennary alpha1,3-fucosylation at sites 3, 4 and 5 were detected. Although there were no significant differences between acute and chronic sera in site directed branching structures, significant differences of alpha1,3-fucosylation were detected in tri-antennary at sites 2, 4 and 5 and in tetra-antennary at sites 3 and 4. CONCLUSION: Little variation in the N-glycan composition of the glycosylation sites of AGP was observed among healthy individuals, while the sera of patients with acute inflammation demonstrated increased numbers of bi-antennary and alpha1,3-fucosylated N-glycan structures at each glycosylation site.     

Identification of a novel apoptosis suppressor gene from the baculovirus Lymantria dispar multicapsid nucleopolyhedrovirus

Ld652Y cells from Lymantria dispar readily undergo apoptosis upon infection with a variety of nucleopolyhedroviruses (NPVs), while L. dispar multicapsid NPV (LdMNPV) infection of Ld652Y cells results in the production of a high titer of progeny viruses. Here, we identify a novel LdMNPV apoptosis suppressor gene, apsup, which functions to suppress apoptosis induced in Ld652Y cells by infection with vAcΔp35, a p35-defective recombinant Autographa californica MNPV. apsup also suppresses apoptosis of Ld652Y cells induced by actinomycin D and UV exposure. Apsup is expressed in LdMNPV-infected Ld652Y cells late in infection, and RNA interference-mediated apsup ablation induces apoptosis of LdMNPV-infected Ld652Y cells.     

N-glycan structures from the major glycoproteins of pigeon egg white: predominance of terminal Galalpha(1)Gal

N-Glycans from major glycoproteins of pigeon egg white (ovotransferrin, ovomucoid, and ovalbumins) were enzymatically released and were reductively aminated with 2-aminopyridine, separated, and structurally characterized by mass spectrometry and a three-dimensional mapping technique using three different columns of high performance liquid chromatography (HPLC) (Takahashi, N., Nakagawa, H., Fujikawa, K., Kawamura, Y., and Tomiya, N. (1995) Anal. Biochem. 226, 139-146). Twenty-five major N-glycan structures, all of them hitherto unknown, were identified as pyridylamino derivatives. Of these, 13 were neutral, 10 were monosialyl, and 2 were disialyl oligosaccharides. All N-glycans contain from one to four Galalpha(1,4)Galbeta(1,4) sequences at the nonreducing terminal positions and are devoid of fucose residues. N-Acetylneuraminic acids were alpha(2,6)-linked only to beta-galactose. The HPLC profiles of the N-glycans from four different glycoproteins were qualitatively very similar to each other, but not identical in the peak distributions. Monosialyl glycans were most abundant in all four glycoproteins, followed by neutral glycans. Disialyl glycans were lowest in ovotransferrin, and highest in ovomucoid. Triantennary structures with bisecting GlcNAc were predominant in ovotransferrin, and tetra-antennary (with and without bisecting GlcNAc-containing) structures were predominant in other glycoproteins. Penta-antennary structures (with a sialic acid and without bisecting GlcNAc residue) were also found in small quantities in all four glycoproteins. In contrast to the chicken egg white counterparts, which contain mostly high mannose and hybrid types, all N-glycan structures in the major pigeon egg white glycoproteins are complex type.     

Engineering Yarrowia lipolytica to produce glycoproteins homogeneously modified with the universal Man3GlcNAc2 N-glycan core

Yarrowia lipolytica is a dimorphic yeast that efficiently secretes various heterologous proteins and is classified as "generally recognized as safe." Therefore, it is an attractive protein production host. However, yeasts modify glycoproteins with non-human high mannose-type N-glycans. These structures reduce the protein half-life in vivo and can be immunogenic in man. Here, we describe how we genetically engineered N-glycan biosynthesis in Yarrowia lipolytica so that it produces Man(3)GlcNAc(2) structures on its glycoproteins. We obtained unprecedented levels of homogeneity of this glycanstructure. This is the ideal starting point for building human-like sugars. Disruption of the ALG3 gene resulted in modification of proteins mainly with Man(5)GlcNAc(2) and GlcMan(5)GlcNAc(2) glycans, and to a lesser extent with Glc(2)Man(5)GlcNAc(2) glycans. To avoid underoccupancy of glycosylation sites, we concomitantly overexpressed ALG6. We also explored several approaches to remove the terminal glucose residues, which hamper further humanization of N-glycosylation; overexpression of the heterodimeric Apergillus niger glucosidase II proved to be the most effective approach. Finally, we overexpressed an α-1,2-mannosidase to obtain Man(3)GlcNAc(2) structures, which are substrates for the synthesis of complex-type glycans. The final Yarrowia lipolytica strain produces proteins glycosylated with the trimannosyl core N-glycan (Man(3)GlcNAc(2)), which is the common core of all complex-type N-glycans. All these glycans can be constructed on the obtained trimannosyl N-glycan using either in vivo or in vitro modification with the appropriate glycosyltransferases. The results demonstrate the high potential of Yarrowia lipolytica to be developed as an efficient expression system for the production of glycoproteins with humanized glycans.     

Occurrence of secretory glycoprotein-specific GalNAc beta 1-->4GlcNAc sequence in N-glycans in MDCK cells

Many reports show that N-glycans of glycoproteins play important roles in vectorial transport in MDCK cells. To assess whether structural differences in N-glycans exist between secretory glycoproteins and membrane glycoproteins, we studied the N-glycan structures of the glycoproteins isolated from MDCK cells. Polarized MDCK cells were metabolically labeled with [3H]glucosamine, and (3)H-labeled N-glycans of four glycoprotein fractions, secretory glycoproteins in apical and basolateral media, and apical and basolateral membrane glycoproteins, were released by glycopeptidase F. The structures of the free N-glycans were comparatively analyzed using various lectin column chromatographies and sequential glycosidase digestion. The four samples commonly contained high-mannose-type glycans and bi- and tri-antennary glycans with a bisected or non-bisected trimannosyl core. However, secretory glycoproteins in both media predominantly contained (sialyl)LacdiNAc sequences, +/-Sia alpha 2-->6GalNAc beta 1-->4GlcNAc beta 1-->R, which linked only to a non-bisected trimannosyl core. beta1-->4N-acetylgalactosaminyltransferase (beta 4GalNAc-T) activity in MDCK cells preferred non-bisected glycans to bisected ones in accordance with the proposed N-glycan structures. This secretory glycoprotein-predominant LacdiNAc sequence was also found in the case of human embryonic kidney 293 cells. These results suggest that the secretory glycoprotein-specific (sialyl)LacdiNAc sequence and the corresponding beta 4GalNAc-T are involved in transport of secretory glycoproteins.     

Glycosylation of site-specific glycans of α1-acid glycoprotein and alterations in acute and chronic inflammation

Backgroundα₁-Acid glycoprotein (AGP), an acute phase reactant, is extensively glycosylated at five Asn-linked glycosylation sites. In a number of pathophysiological states, including inflammation, rheumatoid arthritis, and cancer, alterations of Asn-linked glycans (N-glycans) have been reported. We investigated alteration of N-glycans at each of glycosylation sites of AGP in the sera of patients with acute and chronic inflammation.MethodsAGP purified from sera was digested with Glu-C and the liberated glycopeptides were isolated by reverse phase HPLC. N-glycans released with peptide N-glycosidase F and followed by neuraminidase treatment were analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry.ResultsSite-specific differences in branching structures were observed among N-glycosylation sites 1, 3, 4 and 5. Within the sera of patients with acute inflammation, increases in bi-antennary and decreases in tri- and tetra-antennary structures were observed, as well as increases in α1,3-fucosylation, at most glycosylation sites. In the sera of patients with chronic inflammation, increased rates of tri-antennary α1,3-fucosylation at sites 3 and 4 and tetra-antennary α1,3-fucosylation at sites 3, 4 and 5 were detected. Although there were no significant differences between acute and chronic sera in site directed branching structures, significant differences of α1,3-fucosylation were detected in tri-antennary at sites 2, 4 and 5 and in tetra-antennary at sites 3 and 4.ConclusionLittle variation in the N-glycan composition of the glycosylation sites of AGP was observed among healthy individuals, while the sera of patients with acute inflammation demonstrated increased numbers of bi-antennary and α1,3-fucosylated N-glycan structures at each glycosylation site.     

Analysis of N-glycan in serum glycoproteins from db/db mice and humans with type 2 diabetes

Glycosylation has an important role in regulating properties of proteins and is associated with many diseases. To examine the alteration of serum N-glycans in type 2 diabetes, we used the db/db mouse model. Serum N-glycans were fluorescence labeled and applied to HPLC. There were reproducible differences in N-glycan profiles between the db/db mouse model and the db/+ control. The structures of the oligosaccharides, which had changed in their amounts, were analyzed by a two-dimensional mapping method, matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, and exoglycosidase digestion. Those analyses revealed an increase in the N-glycans possessing alpha1,6-fucose in the serum of db/db mice. The level of alpha1,6-fucosyltransferase mRNA was increased in the liver of the db/db mice. The ratio of a biantennary N-glycan with alpha1,6-fucose to that without alpha1,6-fucose in the liver tissue of the db/db mouse was increased relative to the db/+ control. Next, we analyzed the serum N-glycan profile in human subjects with type 2 diabetes and found an increased amount of a biantennary N-glycan that had an alpha1,6-fucose with a bisecting N-acetylglucosamine. In conclusion, the increase in alpha1,6-fucosylation is a striking change in the serum N-glycans of the db/db mice, whereas the change in the fucosylation in humans with type 2 diabetes was small, albeit statistically significant. It is likely that the change is caused, at least partially, by the increase in the alpha1,6-fucosyltransferase mRNA level in the liver. The increased alpha1,6-fucosylation may affect protein properties associated with the pathophysiology of type 2 diabetes.     

Expression of N-linked sialyl Le(x) determinants and O-glycans in the carbohydrate moiety of human amniotic fluid transferrin during pregnancy

Transferrin, a glycoprotein involved in iron transport in body fluids, was isolated from amniotic fluid of a hydramniospatient by sequential anion-exchange chromatography and gel filtration. The N-glycans of human amniotic fluid transferrin (hAFT) were enzymatically liberated by PNGase-F digestion, isolated by gel filtration and fractionated by (high-pH) anion-exchange chromatography. After alkaline borohydride treatment of native hAFT, the released O-glycans were isolated by gel filtration and fractionated by anion-exchange chroma-tography. Structure elucidation of 14 N- and 2 O-glycans was performed by 500 or 600 MHz1H-NMR spectroscopy. Besides conventional N-glycans established earlier for human serum transferrin (hST), new (alpha1-3)-fucosylated N- glycans were found, representing sialyl Le(x) elements. Furthermore, as compared to hST, a higher degree of (alpha1-6)-fucosylation and an increase in branching from di- to triantennary compounds has been detected. The presence of O-glycans is demonstrated for the first time in transferrin.      

Determination of modulation of ligand properties of synthetic complex-type biantennary N-glycans by introduction of bisecting GlcNAc in silico, in vitro and in vivo.

We have investigated the consequences of introducing a bisecting GlcNAc moiety into biantennary N-glycans. Computational analysis of glycan conformation with prolonged simulation periods in vacuo and in a solvent box revealed two main effects: backfolding of the alpha1-6 arm and stacking of the bisecting GlcNAc and the neighboring Man/GlcNAc residues of both antennae. Chemoenzymatic synthesis produced the bisecting biantennary decasaccharide N-glycan and its alpha2-3(6)-sialylated variants. They were conjugated to BSA to probe the ligand properties of N-glycans with bisecting GlcNAc. To assess affinity alterations in glycan binding to receptors, testing was performed with purified lectins, cultured cells, tissue sections and animals. The panel of lectins, including an adhesion/growth-regulatory galectin, revealed up to a sixfold difference in affinity constants for these neoglycoproteins relative to data on the unsubstituted glycans reported previously [André, S., Unverzagt, C., Kojima, S., Dong, X., Fink, C., Kayser, K. & Gabius, H.-J. (1997) Bioconjugate Chem. 8, 845-855]. The enhanced affinity for galectin-1 is in accord with the increased percentage of cell positivity in cytofluorimetric and histochemical analysis of carbohydrate-dependent binding of labeled neoglycoproteins to cultured tumor cells and routinely processed lung cancer sections. Intravenous injection of iodinated neoglycoproteins carrying galactose-terminated N-glycans into mice revealed the highest uptake in liver and spleen for the bisecting compound compared with the unsubstituted or core-fucosylated N-glycans. Thus, this substitution modulates ligand properties in interactions with lectins, a key finding of this report. Synthetic glycan tailoring provides a versatile approach to the preparation of newly substituted glycans with favorable ligand properties for medical applications.     

Glycosylation of human recombinant gonadotrophins: characterization and batch-to-batch consistency

The glycan moiety of human recombinant gonadotrophins (r-hFSH, r-hLH, and r-hCG) produced in CHO cell lines has been characterized by a combination of chromatographic and mass spectrometric techniques, including both matrix-assisted laser desorption ionization and electrospray. Two glycan mapping methods have been developed for the three gonadotrophins that allow separation of the glycans according to either their charge or sialylation level or their antennarity. A method was also developed for r-hCG that permits the complete resolution of the N-glycan from the O-glycan species. Whereas the structure found for the N-glycans of the gonadotrophins was in agreement with the complex type model, the structure for an O-glycan of r-hCG, not yet described, has been unambiguously determined using nanoelectrospray ion trap mass spectrometry. Using these two glycan mapping methods, the high level of batch-to-batch consistency achieved for the glycosylation of the three recombinant gonadotrophins in commercial production has been shown. These data demonstrate the tight control that can be achieved in the manufacturing of complex recombinant therapeutic glycoproteins, which is a prerequisite to the delivering of a guaranteed dose of drug from vial to vial, and in turn to ensuring the clinical efficacy of the product.